Project Summary During development of the vertebrate nervous system, a vast array of neurons will develop in discrete anatomical positions, acquire varied morphological forms, and establish connections with specific populations of target cells. Such spatial organization of cell fates and differentiation during the development of the nervous system are directed by concentration gradients of chemical signals, termed morphogens. Even though the importance of graded morphogen signaling in developmental pattern formation has been well recognized, it remains a significant question in biology about how embryonic progenitor cells transform dynamic changes in developmental signaling into spatial patterns of gene expression and cellular differentiation in a reliable and robust fashion. The long-term functional goal of this NIH R21 project is to specifically address the significant challenge in understanding the interpretation of morphogen gradients by intracellular signaling cascades while embryonic precursor cells are undergoing multicellular self-organization during developmental patterning. Specifically, we propose to leverage the intrinsic lumenogenic and self-organizing properties of neuroepithelial (NE) cells, the embryonic precursor cells in the neural tube, in conjunction with an innovative microfluidic embryological device, to achieve controllable and reproducible generations of lumenal NE cysts to mimic un- patterned spinal cord tissues. High-purity NE cells will be derived from human pluripotent stem cells (hPSCs) using established 2D directed differentiation protocols. Lumenal NE cysts will then be utilized seamlessly in the same microfluidic device for downstream asymmetrical patterning using the morphogen Sonic hedgehog (Shh) to achieve progressive acquisition of ventral neuronal subtypes in the spinal cord. Successful accomplishment of this proposed research will lead to the establishment of an innovative microfluidics-based methodology for controllable, reproducible, and scalable generation of (autologous) human spinal cord tissues from hPSCs, a quantum leap compared with existing 3D organoid culture systems that are known to lack controllability and reproducibility. Furthermore, our synthetic patterned human spinal cord model will provide a very useful experimental platform that offers superior experimental controls of key parameters and quantitative measurements to allow in-depth mechanistic investigations on the emergent self-organizing principles and pattering mechanisms that provide robustness and reliability to embryonic patterning, a long-standing question in biology.